January 2009
Volume 23 • Number 1
ORIGINAL
RESEARCH
GNRH Induces the Unfolded Protein Response in the
L␤T2 Pituitary Gonadotrope Cell Line
Minh-Ha T. Do, Sharon J. Santos, and Mark A. Lawson
Department of Reproductive Medicine (M.-H.T.D., S.J.S., M.A.L.) and Biomedical Sciences Graduate Program (M.H.T.D), University of California, San Diego, La Jolla, California 92093
The neuropeptide GNRH 1 stimulates the secretion of the reproductive hormone LH in pituitary
gonadotropes. Other secretory cell types depend on the unfolded protein response (UPR) pathway to regulate protein synthesis and protect against endoplasmic reticulum (ER) stress in response to differentiation or secretory stimuli. This study investigated the role of the UPR in GNRH
action within the L␤T2 gonadotrope model. Cells were treated with GNRH, and the activation of
UPR signaling components and general translational status was examined. The ER-resident stress
sensors, Atf6, Eif2ak3, and Ern1, are all present, and GNRH stimulation results in the phosphorylation of eukaryotic translation initiation factor 2A kinase 3 and its downstream effector, eukaryotic translation initiation factor 2A. Additionally, activation of the UPR was confirmed both in
L␤T2 as well as mouse primary pituitary cells through identifying GNRH-induced splicing of Xbp1
mRNA, a transcription factor activated by splicing by the ER stress sensor, ER to nucleus signaling
1. Ribosome profiling revealed that GNRH stimulation caused a transient attenuation in translation, a hallmark of the UPR, remodeling ribosomes from actively translating polysomes to translationally inefficient ribonucleoprotein complexes and monosomes. The transient attenuation of
specific mRNAs was also observed. Overall, the results show that GNRH activates components of
the UPR pathway, and this pathway may play an important physiological role in adapting the ER
of gonadotropes to the burden of their secretory demand. (Molecular Endocrinology 23: 100 –112,
2009)
T
he reproductive axis is controlled by release of the decapeptide GNRH 1 (also LHRH) from the hypothalamus. GNRH
binding to its G protein-coupled receptor on gonadotrope cells
in the anterior pituitary activates and maintains the synthesis of
gonadotropin hormones LH and FSH and stimulates release of
preformed stores of LH (1). These hormones, in turn, act on the
gonad to regulate folliculogenesis, ovulation, spermatogenesis,
and steroidogenesis. LH and FSH are glycoprotein heterodimers, each comprised of a common ␣-glycoprotein subunit
(Cga, also ␣GSU) and a unique ␤-subunit (Lhb or Fshb) (2).
Studies in pituitary gonadotropes are made possible by the
L␤T2 cell model, created through immortalization of mouse
gonadotropes by targeted tumorigenesis (3). L␤T2 cells have
been shown to respond to GNRH by raising intracellular
calcium levels and stimulating exocytosis (4). The signal
transduction pathways and transcriptional responses induced
by GNRH, including induction of Lhb and Cga among other
genes important to gonadotrope function, have been extensively characterized (2, 5) in part by utilizing this and other
gonadotrope models (6).
Whereas the transcriptional regulation is well described, very
little work has focused on posttranscriptional regulatory pathways induced by GNRH. One important posttranscriptional
pathway is the unfolded protein response (UPR), which incorporates both transcriptional and posttranscriptional mechanisms in a multifaceted response to minimize endoplasmic reticulum (ER) stress. The ER lumen is an oxidative environment
where protein folding and posttranslational modification of
ISSN Print 0888-8809 ISSN Online 1944-9917
Printed in U.S.A.
Copyright © 2009 by The Endocrine Society
doi: 10.1210/me.2008-0071 Received March 3, 2008. Accepted October 23, 2008.
First Published Online October 30, 2008
Abbreviations: 2-APB, 2-Aminoethoxydiphenyl borate; ATF6, activating transcription factor 6; BAPTA, 1,2-bis(o-aminophenoxy)ethane-N,N,N⬘,N⬘-tetraacetic acid; DTT, dithiothreitol; EGF, epidermal growth factor; EIF2A, eukaryotic translation initiation factor 2A;
EIF2AK3, EIF2A kinase 3; ER, endoplasmic reticulum; ERN1, ER to nucleus signaling 1;
ERSE, ER stress response element; FRAP, FK506-binding protein 12-rapamycin associated
protein 1; fwd, forward; HSPA5, heat shock 70-kDa protein 5; IP3, inositol 1,4,5 triphosphate; PEB, polysome extraction buffer; PI3K, phosphatidylinositol 3-kinase; PKC, protein
kinase C; rev, reverse; RNP, ribonucleoprotein; ROS, reactive oxygen species; UPR, unfolded protein response.
100
mend.endojournals.org
Mol Endocrinol, January 2009, 23(1):100 –112
Mol Endocrinol, January 2009, 23(1):100 –112
proteins that are secreted or targeted to the membranes occurs. The
UPR is a quality control pathway that monitors changes in the ER
lumen that perturb protein-folding capacity. Disruption of the ER
lumen can be a result of pathological conditions such as hypoxia,
viral infection, starvation, or a result of normal physiological processes such as secretion or high-protein synthetic demand. ER stress
can also be induced experimentally by the overexpression of misfolded proteins, which overwhelm the ER, or by pharmacological
insults that target glycosylation, calcium, or oxidative balance. The
UPR seeks to reestablish balance and decrease burden by attenuating translation and degrading misfolded proteins, as well as increasing synthetic capacity by increasing the size of the ER and the
capacity of the protein-folding machinery. If balance is not
reached, the UPR induces apoptosis.
Stress in the ER lumen is sensed by three ER-resident proteins, EIF2A kinase 3 (EIF2AK3, also known as PERK or PEK),
ER to nucleus signaling 1 (ERN1, also known as IRE1), and
activating transcription factor 6 (ATF6). Activation of EIF2AK3
leads to an immediate attenuation of general translation
through phosphorylation of eukaryotic translation initiation
factor 2A (EIF2A, also known as eIF2␣), which reduces protein
synthesis demand on the ER. Phosphorylation of EIF2A also
causes translation stimulation of Atf4 (activating transcription
factor 4), a bZIP transcription factor that stimulates genes that
further the UPR program, including those involved in amino
acid transport, synthesis, metabolism, and the antioxidant response. EIF2AK3 activation is followed temporally by proteolytic activation of the basic leucine-zipper transcription factor
ATF6, which regulates genes with ER stress response elements
(ERSEs), such as chaperones. Finally, the UPR activates the
kinase/endoribonuclease ERN1, which splices Xbp1 (X-box
binding protein 1) mRNA, another bZIP transcription factor
important for UPR transcriptional responses. XBP1 acts on promoters at UPR elements and is thought to be responsible for
regulating genes that mediate ER-associated degradation of misfolded proteins. The UPR signaling pathway has been studied
extensively and reviewed recently (7–10). Another, recently discovered arm of the stress response is inhibition of translational
complexes at the ER translocon (11).
Recently, the UPR was shown to be crucial for the function of
secretory cells, including plasma cells, ␤-cells, hepatocytes, and
osteoblasts (12), all of which have heavy protein synthesis demands and thus rely on the proper function of the ER to maintain secretory output. Loss of Xbp1 in B lymphocytes in mice
results in failure to differentiate into immunoglobulin-secreting
plasma cells and ultimately failure to mount an immune response to polyoma virus infection (13). Ern1 is also required for
proper immunoglobulin production and plasma cell differentiation (14). Mice that lack functional EIF2AK3 in insulin-secreting pancreatic ␤-cells have elevated serum glucose levels compared with wild-type littermates and eventually experience
␤-cell apoptosis and diabetes (15, 16). Transgenic mice harboring a mutation in Eif2a that does not allow phosphorylation
show impaired insulin production and loss of insulin-positive
␤-cells. Most transgenic neonates die within 18 h of birth (17).
Because gonadotropes are endocrine secretory cells that experience heavy secretion and protein synthesis demand, it can be
mend.endojournals.org
101
hypothesized that the UPR plays a role in regulating function in
these cells. The UPR is currently unexplored in gonadotropes,
although indirect evidence indicates that this may be the case.
Microarray studies have shown that GNRH induces Atf3 (18 –
20), a transcription factor induced by the EIF2AK3/ATF4 arm
of the UPR (21). GNRH has been shown to transcriptionally
regulate genes involved in amino acid synthesis, metabolism,
and oxidative stress (18, 19), also consistent with activation of
EIF2AK3. Finally, Xbp1 predicted that targets such as those of
the DnaJ family (22) are induced by GNRH (19, 20). To meet
the demands of hormone secretion, it is likely that gonadotropes
mount a UPR-like response to GNRH. This hypothesis is addressed in this study by examiniation of the activation of UPR
effectors in the L␤T2 gonadotrope cell line and primary pituitary gonadotropes and by ribosome profiling to examine the
general status of translation. The results indicate that the UPR is
a target of GNRH action within the gonadotrope.
Results
GNRH activates the ER stress sensor EIF2AK3
The main sensors of ER stress are three ER-resident proteins,
ERN1, EIF2AK3, and ATF6. RT-PCR of mRNA isolated from
L␤T2 cells indicates that all three sensors are expressed in the
cell line (Fig. 1A). An immediate effect of ER stress is activation
of the UPR through EIF2AK3. Activated EIF2AK3 oligomerizes
(23) and autophosphorylates at 10 different sites in its kinase
domain (24). To determine whether GNRH exposure leads to
the phosphorylation of EIF2AK3, L␤T2 cells were treated with
GNRH or dithiothreitol (DTT) for 30 min. DTT severely disrupts the ER’s oxidative environment required for proper protein folding (10) and is commonly used to disrupt translation
and activate the UPR (11, 25, 26). Protein was harvested and
subjected to Western blotting. A shift in EIF2AK3 mobility (Fig.
1B) is consistent with hyperphosphorylation of the protein, with
DTT causing a slightly greater shift than seen with GNRH. To
confirm that this shift is indeed due to phosphorylation events,
protein extracts were treated with phosphatase and then subjected to Western blotting. The mobility shift of EIF2AK3 is
abrogated by phosphatase treatment of extracts (Fig. 1C). Under basal conditions EIF2AK3 exists in a moderately phosphorylated state, with the level of phosphorylation increasing with
GNRH or DTT treatment.
Activated EIF2AK3 directly phosphorylates EIF2A, which
is the ␣-subunit of the eIF2 translation initiation complex.
Phosphorylation of EIF2A prevents initiation complex formation, leading to free ribosomal subunit accumulation and
a decrease in general translation (27). To examine the phosphorylation status of EIF2A in response to GNRH, antibodies specific to total and phosphorylated EIF2A were used in
Western blotting of GNRH and DTT-treated extracts.
GNRH treatment resulted in an increase in EIF2A phosphorylation, and greater phosphorylation was observed after DTT
treatment (Fig. 1D).
102
Do et al.
GNRH Induces the UPR
A
Eif2ak3
RT:
Mol Endocrinol, January 2009, 23(1):100 –112
Atf6
+ - +
-
A
Ern1
+
-
LβT2 Total Xbp1 RT-PCR
200
RT:
Vehicle
GNRH
+
+
-
DTT
-
+
-
150
100
B
200
Total EIF2AK3
DTT
PstI digestion of Xbp1
GNRH
MW
Vehicle
DTT
B
GNRH
Vehicle
550
140
Spliced
Xbp1
550
C
Phosphatase:
Vehicle
GNRH
-
-
+
+
DTT
-
+
Unspliced
Xbp1
P-EIF2AK3
EIF2AK3
D
400
200
Vehicle
GNRH
DTT
C
6.0
Primary pituitary cells
Spliced Xbp1
Total EIF2A
FIG. 1. Expression of ER stress sensors and activation of EIF2AK3 by GNRH in
L␤T2 gonadotropes. A, Total RNA was isolated from untreated cells and PCR was
performed using transcript-specific primers to determine presence of the three
known ER stress sensors Eif2ak3, Atf6, and Ern1. RT, Reverse transcriptase. B,
Total protein was harvested from L␤T2 cells treated with vehicle, 10 nM GNRH, or
2 mM DTT for 30 min and subjected to Western blotting. Antibody against total
EIF2AK3 was used to detect EIF2AK3 in lysates. Hyperphosphorylation of
EIF2AK3 is indicated by decreased electrophoretic mobility. C, The
phosphorylation-dependent decrease in EIF2AK3 mobility with GNRH or DTT
treatment was confirmed by treatment of protein extracts with ␭-protein
phosphatase before Western blotting. D, Phosphorylation of the EIF2AK3 target
EIF2A was confirmed by Western blotting with antibodies specific to the
phosphorylated and total forms of the protein. MW, Molecular weight.
GNRH causes splicing of the UPR transcription
factor Xbp1
To confirm that GNRH is inducing ER stress, a different arm
of the UPR was explored. Signaling to ERN1 leads to activation
of Xbp1, which encodes a bZIP transcription factor that acts at
UPR elements (8). Xbp1 is activated through a cytoplasmic
mRNA splicing event, where removal of a small intron allows
translation of an active transcription factor (28). The unspliced
mRNA contains a PstI site in the intron that is removed by the
splicing event (29). Xbp1 cDNA prepared from L␤T2 cells
treated with vehicle, GNRH, or DTT (Fig. 2A) was subjected to
PstI digestion. GNRH caused splicing of Xbp1 mRNA, although to a lesser degree than DTT treatment (Fig. 2B), which
resulted in splicing of all the available Xbp1 mRNA.
To confirm that the UPR is activated by GNRH in primary
gonadotrope cells, pituitaries were isolated from wild-type, sexually mature 9-wk-old male mice, dissociated, and cultured.
Only gonadotropes express the receptor for GNRH (30) and
thus should be the only cell type responding to GNRH. The cells
Xbp1s/Gapdh
P-EIF2A
*
4.0
2.0
0.0
Vehicle
GNRH
FIG. 2. GNRH causes splicing of the UPR transcription factor Xbp1 mRNA in
L␤T2 and mouse primary gonadotropes. A, L␤T2 cells were treated with GNRH
or DTT for 30 min and total RNA was harvested. Xbp1 mRNA was reverse
transcribed and amplified using gene-specific primers. The resulting cDNA was
subjected to agarose electrophoresis to confirm appropriate amplicon size. RT,
Reverse transcriptase. B, Amplified Xbp1 cDNA was digested with PstI to confirm
the loss of a PstI restriction site within the intron sequence. Spliced Xbp1 cDNA is
insensitive to PstI digestion. C, Primary pituitary cells cultured from wild-type, 9wk-old male mice were plated and treated with GNRH or DTT for 30 min. RNA
was isolated and quantitative PCR was used to measure the spliced form of
Xbp1 specifically (Xbp1s). Gapdh was used as an internal control. The
reported values are the means ⫾ SEM of three independent experiments.
Asterisks indicate statistical significance from a value of 1 as determined by
Student’s t test (P ⱕ 0.05).
were treated with GNRH for 30 min, and the splicing of Xbp1
was measured through quantitative PCR using primers specific
to the spliced form of Xbp1 (Xbp1s). Splicing of Xbp1 increased
4-fold in response to GNRH (Fig. 2C).
GNRH causes an accumulation of ribonucleoprotein
(RNP) complexes
The observation that GNRH causes EIF2A phosphorylation
(Fig. 1D) indicates that GNRH may attenuate translation. To
determine the general translational status of the L␤T2 cells, the
cells were treated with GNRH for 30 min, and then ribosome
and mRNA complexes were separated on sucrose gradients.
Complexes were fractionated according to density while monitoring absorption at 254 nm. Presence of the 28S and 18S rRNAs was used to facilitate combining fractions to represent the
Mol Endocrinol, January 2009, 23(1):100 –112
mend.endojournals.org
actively translating polysome pool, consisting of mRNAs complexed with two or more ribosomes, or the RNP pool, consisting
of mRNAs complexed with initiation factors or a single ribosome (monosome), and free 60S or 40S ribosomal subunits (Fig.
3A). The relative abundance of RNA in each pool was compared
using the absorption profiles to evaluate the extent of translational regulation by GNRH. This comparison indicated a
A
OD 254 nm
5
Polysomes
4
RNP
60S
80S 40S
3
2
103
marked redistribution of RNA in response to acute GNRH,
reflecting a general shift from actively translating polysomes to
RNP complexes (Fig. 3B). The areas under the ribosome profile
curves of the pools were integrated as a measure of the amount
of RNA present in each pool. The RNP pool shifted from being
about 60% of the polysome pool to being equivalent to the
polysome pool with GNRH exposure (Fig. 3C). The degree of
remodeling was dose dependent when comparing concentrations of 1, 10, and 100 nM (data not shown).
As a comparison, cells were treated with epidermal growth
factor (EGF), a mitogen known to stimulate translation (31) but
not induce the UPR. As expected, an increase in polysomes
compared with RNP was observed (Fig. 3B). In contrast, treatment of the cells with DTT resulted in the expected depletion of
polysomes (Fig. 3B), similar in direction but greater in magnitude than that of GNRH.
1
0
28S rRNA
18S rRNA
B
Vehicle
GNRH
RNP
Polysomes
RNP
Polysomes
EGF
DTT
RNP
Polysomes
RNP
Polysomes
C
B
RNP/Polysome
Area Ratio
1.25
1.0
0.75
A
0.5
0.25
0.0
Vehicle
GNRH
FIG. 3. GNRH exposure causes an accumulation of RNP complexes in L␤T2 cells.
A, Sample ribosomal profile from untreated L␤T2 cells. RNA and ribosome
complexes were harvested and fractionated on sucrose gradients while
monitoring UV absorption at 254 nm. Fractions were pooled to represent
polysomes (RNA with two or more ribosomes) and RNP complexes (ribosomal
subunits, monosomes, initiation complexes), as shown. The peaks in the RNP
fractions correspond to the individual ribosomal subunits and full 80S
monosomes, as indicated by the relative proportions of 28S and 18S rRNA,
representing the presence of the large and small ribosomal subunits, respectively.
B, Representative ribosomal profiles of cells treated with GNRH, EGF, or DTT for
30 min. C, The ratio (RNP/polysome) of the integrated area under the curve from
the profiles, as an indication of the level of redistribution of ribosomes. The
reported values are the means ⫾ SEM of six independent experiments. Groups not
connected by the same letter are significantly different (P ⱕ 0.05) as determined
by ANOVA and post hoc Tukey’s HSD test.
Disruption of intracellular calcium mimicks RNP
accumulation by GNRH
GNRH is responsible for inducing secretion and de novo
synthesis of gonadotropins in gonadotropes. GNRH mainly
acts through a G protein-coupled receptor to activate G␣q and
phospholipase C-␤, mediating production of inositol 1,4,5
triphosphate (IP3) and diacylglycerol signaling intermediates.
Diacylglycerol mediates activation of protein kinase C (PKC) (␧,
␦) and all four MAPK cascades, ERK, c-Jun N-terminal kinase,
p38, and MAPK7 (also known as BMK1) (5). ERK has been
implicated in regulation of translation by GNRH (32). The IP3mediated release of calcium from internal stores triggers secretion, whereas calcium influx from the extracellular environment
renews internal stores (5). Modulation of protein synthesis in
response to mitogenic signals is often regulated through phosphatidylinositol 3-kinase (PI3K) and FRAP (FK506-binding
protein 12-rapamycin associated protein 1, also known as
MTOR), the downstream targets of which include 4E-binding
protein (EIF4EBP1, also known as 4EBP1 or PHAS-1) and ribosomal protein S6 kinase (p70s6k) (33). FRAP is a target of
receptor tyrosine kinases and G protein-coupled receptors (34).
To understand the effect on translation mediated by GNRH,
L␤T2 cells were treated with pharmacological inhibitors of the
various signaling pathways before GNRH exposure to determine which signaling pathways are required. The use of U0126
(Fig. 4B) to block ERK activation had no effect on GNRHinduced RNP accumulation. The efficacy of U0126 was confirmed by Western blotting for the phosphorylation status of
ERK after GNRH treatment, in the presence of U0126 (Fig. 4C).
Similar results were obtained using PD98059 to block ERK,
SB203580 to block p38, SP600125 to block c-Jun N-terminal
kinase (data not shown), or bisindolylmaleimide I to block PKC
activation (supplemental data published as supplemental Fig. 1
on The Endocrine Society’s Journals Online web site at http://
mend.endojournals.org). The results indicate that the PKC and
MAPK signaling pathways are not required for ribosomal
remodeling.
Similar to the inhibition of PKC/ERK, inhibiting calcium/
calmodulin-dependent protein kinase II ␣ activation via KN62
and voltage-gated calcium channel activation via nimodipine
104
Do et al.
GNRH Induces the UPR
Mol Endocrinol, January 2009, 23(1):100 –112
A
Vehicle
GNRH
B
U0126
U0126
+ GNRH
Bisindolylmaleimide I
Bisindolylmaleimide I
+ GNRH
C
Vehicle GNRH
U0126
+GNRH
Western Blot
Phospho-ERK
D
2-APB
Thapsigargin
(35), signaling through PI3K and/or FRAP would result in a
positive effect on translation, which is not consistent with an
accumulation of RNP complexes. In L␤T2 cells, PI3K appears
to diverge from the classical receptor tyrosine kinase-signaling
cascade, not utilizing AKT1 (and presumably, then, FRAP) in its
direct downstream effects on cell survival (35), gonadotropin
synthesis (36), or translation initiation factors (32). For these
reasons, the PI3K/FRAP arm was not pursued further.
In contrast, ER calcium disrupters such as thapsigargin,
which blocks calcium reuptake by the sarcoplasmic reticulum/ER Ca2⫹ adenosine triphosphatase pump, 2-aminoethoxydiphenyl borate (2-APB), which blocks IP3 receptors, calcium
ionophore ionomycin, which increases intracellular calcium levels and is a known LH secretagogue (37), and 1,2-bis(o-aminophenoxy)ethane-N,N,N⬘,N⬘-tetraacetic acid (BAPTA), which
chelates calcium, were all sufficient to induce RNP remodeling,
similar to GNRH (Fig. 4, D and E). Pharmacological agents that
disrupt ER calcium stores have been shown to induce the UPR
(38). Thus, ER and intracellular calcium levels appear to be
involved in mediating RNP accumulation in response to GNRH
stimulation.
Ionomycin
BAPTA
B
Area Ratio
RNP/Polysome
1.5
B
5
GNRH
4
3
2
B
1.0
0.5
A
0.0
Vehicle GNRH Ionomycin
FIG. 4. Accumulation of RNP complexes is mimicked by disruption of
intracellular calcium. L␤T2 cells were treated and ribosomes were fractionated
while monitoring UV absorption. A, Representative profiles from cells treated
with vehicle or GNRH for 30 min. B, Representative profiles from cells treated
with U0126 to inhibit MEK/ERK activation, before GNRH exposure for 30 min. C,
The efficacy of U0126 was confirmed through Western blotting to determine
ERK phosphorylation status. D, Representative profiles of cells treated with
sarcoplasmic reticulum/ER Ca2⫹ adenosine triphosphatase antagonist
thapsigargin, IP3-receptor antagonist 2-APB, calcium ionophore, and
secretagogue ionomycin, or calcium chelator BAPTA. The profiles indicate that
disruption of calcium homeostasis causes an accumulation of RNP complexes
similar to GNRH. E, The ratio (RNP/polysome) of the integrated area under the
curve from the profiles of GNRH and ionomycin-treated cells, as an indication of
the level of redistribution of RNA. The reported values are the means ⫾ SEM of four
independent experiments. Groups not connected by the same letter are significantly
different (P ⱕ 0.05) as determined by ANOVA and post hoc Tukey’s HSD test.
had no effect (data not shown). The use of translation inhibitors
LY 294002 to block PI3K or rapamycin to block FRAP were not
fruitful, because these agents alone caused translation attenuation (data not shown). Furthermore, although GNRH has been
shown to activate PI3K via transactivation of the EGF receptor
*
Ionomycin
* *
*
*
1
Lhb
Fold-Redistribution to RNP
E
Fold-Redistribution to RNP
A
Cga
Gapdh
*
25
GNRH
20
Dithiothreitol
*
15
10
5
1
*
*
*
Lhb
Cga
Gapdh
FIG. 5. Lhb, Cga and Gapdh mRNAs redistribute to RNP complexes in
response to GNRH. L␤T2 cells were treated as indicated, and ribosome/RNA
complexes were fractionated. mRNA was isolated from the RNP and polysome
pools of the fractionated extracts and measured using quantitative PCR. A
fold-redistribution value was calculated as a measurement of the change in
transcript representation in the pools after treatment, as compared with
control (vehicle) treatment. The data are represented in the histogram such
that a positive value indicates movement into the RNP pool, and a negative
fold-redistribution value indicates movement into the polysome pool. A foldredistribution value of 1 represents no change after treatment. A, The
redistribution of mRNAs was calculated for cells exposed to 30 min of GNRH
(black) or ionomycin (white). The reported values are the means ⫾ SEM of four
independent experiments. B, The redistribution of mRNAs was calculated for
cells exposed to 30 min of GNRH (black) or DTT (white). The reported values
are the means ⫾ SEM of three independent experiments. Asterisks indicate
statistical significance from a value of 1 as determined by Student’s t test
(P ⱕ 0.05).
Mol Endocrinol, January 2009, 23(1):100 –112
mend.endojournals.org
Lhb, Cga, and Gapdh mRNAs redistribute to
RNP complexes
To evaluate whether the overall ribosome changes seen after
exposure to GNRH are reflected in the manner in which specific
mRNAs relevant to gonadotrope function behave, Lhb and Cga
mRNAs were examined. In the differentiated gonadotrope,
GNRH stimulates transcription and synthesis of the LH subunits, which are modified and exported through the ER and are
therefore dependent on proper ER function.
The behavior of Lhb and Cga mRNA was determined by
measuring their mass ratio between the polysome and RNP
pools. Redistribution of an mRNA species was calculated by
comparing these ratios in GNRH-treated and vehicle-treated
(control) samples. The calculated fold redistribution is thus a
measurement of the change in transcript representation in each
of the pools after GNRH treatment. In agreement with the overall shift of ribosomes (Figs. 3 and 4), Lhb, Cga, and Gapdh
mRNAs all redistributed to RNP complexes after GNRH exposure (Fig. 5). This behavior was recapitulated by ionomycin (Fig.
5A) and DTT (Fig. 5B) treatment. The magnitude of redistribution of ionomycin mimicked GNRH, whereas DTT was much
greater, consistent with the degree of ribosome movement induced by these agents (Figs. 3 and 4).
B
Vehicle,
Vehicle
Vehicle,
GNRH
GnRH
OD 254 nm
AD = Actinomycin D
D
Actinomycin D,
Vehicle
AD
Actinomycin D,
GNRH
AD +
GnRH
Area Ratio
RNP/Polysome
2.0
C
1.5
1.0
B
A
0.5
0.0
AD
GnRH
C
OD 254 nm
GNRH attenuates translation initiation
The observed accumulation of RNP complexes after treatment
with GNRH may be a result of two distinct responses. First, the
stimulatory effect of GNRH on transcription of various genes has
been well described (18 –20, 39, 40) and so GNRH could be stimulating production of new mRNA and ribosomes that are assembling in new translation initiation complexes, resulting in an increase of RNP complexes. In this scenario, inhibition of
transcription would block any GNRH-induced redistribution. To
test this possibility, L␤T2 cells were treated with actinomycin D to
block transcription before treatment with GNRH, and ribosomes
were fractionated. Actinomycin D alone caused a slight redistribution of RNA into the RNP pool (Fig. 6, A and B). However, GNRH
caused an additional influx, similar in magnitude to that seen in
control samples. Measurement of the distribution of Lhb, Cga, and
Gapdh mRNA was also consistent with this behavior (Fig. 7A).
The efficacy of actinomycin D was confirmed by its ability to inhibit
stimulation of Egr1 transcription by GNRH (data not shown). Because actinomycin D did not block the remodeling, this indicates that
the rise in RNP complexes and accumulation of mRNAs within those
complexes are independent of transcriptional events.
The second cause of RNP accumulation may be ribosome
dissociation from polysomal mRNA after exposure to GNRH,
-
+
+
-
+
+
D
Vehicle,
Vehicle
Vehicle,
GNRH
GnRH
1.5
CX = Cycloheximide
C
Cycloheximide,
Vehicle
CX
Cycloheximide,
GNRH
CX +
GnRH
Area Ratio
RNP/Polysome
OD 254 nm
OD 254 nm
A
105
1.0
B
B
+
-
+
+
A
0.5
0.0
CX
GnRH
-
+
FIG. 6. GNRH attenuates translation by redistributing ribosomes to RNP complexes. L␤T2 cells were pretreated with actinomycin D (AD) to block transcription or
cycloheximide (CX) to block translation elongation and then treated with 10 nM GNRH or vehicle (PBS) for 30 min. Ribosome/RNA complexes were fractionated while
monitoring UV absorption. A and C, Representative profiles. B and D, The ratio (RNP/polysome) of the integrated area under the curve from the profiles, as an
indication of the level of redistribution of ribosomes. Cycloheximide, but not actinomycin D, blocks redistribution in response to GNRH, indicating that active
translation, but not new mRNA or ribosome synthesis, is necessary for the accumulation of RNP complexes. The reported values are the means ⫾ SEM of four or more
independent experiments. Groups not connected by the same letter are significantly different (P ⱕ 0.05) as determined by ANOVA and post hoc Tukey’s HSD test.
Do et al.
5
4
3
2
Mol Endocrinol, January 2009, 23(1):100 –112
GNRH
*
Actinomycin D + GNRH
*
*
*
*
*
1
Lhb
Gapdh
3.0
2.5
2.0
*
*
*
1.5
Cga
Gapdh
Fold-Redistribution
to polysomes
1.0
A
1.0
C
Lhb
0.8
-1.5
RNP/Polysome
Area Ratio
Fold-Redistribution
to RNP
B
Cga
Attenuation of translation is transient
The primary function of pituitary gonadotropes is to produce LH and FSH, and thus it seems paradoxical that GNRH
would decrease the translation of mRNAs that are important for
the production of these hormones. It was of interest to determine whether this attenuation is permanent or transient. To test
this, L␤T2 cells were exposed to a 10-min pulse of GNRH and
fractionated at various times after stimulation. Integration of
the areas under curve of the resulting ribosome profiles indicates
that a 10-min exposure to GNRH is sufficient to cause an accumulation of RNP complexes similar in magnitude to a 30-min
exposure (Fig. 8). After 60 min, redistribution into RNP complexes subsides, but does not return to basal levels (Fig. 8A).
Using quantitative PCR to follow the distribution of Lhb, Cga,
and Gapdh mRNAs between polysomes and RNP complexes
revealed that whereas the RNP accumulation does not com-
GNRH
-2.0
Cycloheximide + GNRH
FIG. 7. Translation of Lhb, Cga, and Gapdh mRNAs is attenuated. L␤T2 cells
were pretreated with actinomycin D to block transcription or cycloheximide to
block translation elongation and then treated with 10 nM GNRH or vehicle (PBS)
for 30 min. Ribosome/RNA complexes were fractionated. mRNA was isolated
from the RNP and polysome pools of the fractionated extracts and measured
using quantitative PCR. A fold-redistribution value was calculated as a
measurement of the change in transcript representation in the pools after
treatment, as compared with control (vehicle) treatment. The data are
represented in the histogram such that a positive value indicates movement into
the RNP pool, and a negative value indicates movement into the polysome pool.
A fold-redistribution value of 1 represents no change after treatment. A, The
redistribution of mRNAs was calculated for cells exposed to 30 min of GNRH
(black) or pretreated with actinomycin D before GNRH (white). B, The
redistribution of mRNAs was calculated for cells exposed to 30 min of GNRH
(black) or pretreated with cycloheximide before GNRH (white). All the reported
values are the means ⫾ SEM of four or more independent experiments. Asterisks
indicate statistical significance from a value of 1 as determined by Student’s t test
(P ⱕ 0.05).
thus redistributing mRNAs into the RNP pool, such as predicted
by activation of the UPR and an attenuation of translation. In
this scenario, inhibition of translation elongation would prevent
ribosomes from dissociating, thus blocking any GNRH effect.
To test this, L␤T2 cells were pretreated with cycloheximide to
block translation elongation before treatment with GNRH.
Blocking translation elongation indeed blocked the redistribution of ribosomes (Fig. 6, C and D) as well as the redistribution
of Lhb, Cga, and Gapdh mRNAs (Fig. 7B). Cycloheximide pretreatment alone caused a slight change in the distribution of
RNA (Fig. 6, C and D). However, GNRH did not cause a further
redistribution. Additionally, no changes in total mass of RNA
were detected between vehicle and GNRH-treated cells, consistent with ribosome redistribution rather than influx of newly
synthesized mRNA and ribosomes (data not shown). Overall,
the results confirm that the RNP increase caused by GNRH is
indicative of an attenuation of translation.
B
0.6
0.4
A
0.2
0.0
Vehicle
B
Fold-Redistribution
to RNP
Fold-Redistribution
to RNP
A
GNRH Induces the UPR
30 min
*
3.0
2.5
2.0
1.5
60 min
*
*
Cga
1.0
Fold-Redistribution
to polysomes
106
Lhb
Gapdh
-1.5
-2.0
30 min
60 min
FIG. 8. Attenuation of translation by GNRH is transient. L␤T2 cells were
treated with GNRH for 10 min and then GNRH was removed, cells were
rinsed, and the media were replaced by fresh media. The cells were incubated
for another 20 min or 50 min before harvest. Ribosome/RNA complexes were
then fractionated. A, The ratio (RNP/polysome) of the integrated area under
the curve from the profiles, as an indication of the level of redistribution of
ribosomes. B, mRNA from the RNP and polysome pools of fractionated
extracts was measured using quantitative PCR. A fold-redistribution value was
calculated as a measurement of the change in transcript representation in the
pools after GNRH treatment, as compared with control (vehicle) treatment.
The data are represented in the histogram such that a positive value indicates
movement into the RNP pool. A fold-redistribution value of 1 represents no
change. The reported values are the means ⫾ SEM of six independent
experiments. Groups not connected by the same letter are significantly
different (P ⱕ 0.05) as determined by ANOVA and post hoc Tukey’s HSD test.
Asterisks indicate statistical significance from a value of 1 as determined by
Student’s t test (P ⱕ 0.05).
Mol Endocrinol, January 2009, 23(1):100 –112
pletely resolve, the distribution of the specific transcripts examined does return to basal levels within 60 min (Fig. 8B).
mend.endojournals.org
GNRH
GNRHR
1.
PLCβ
Discussion
The data show that in pituitary gonadotropes, GNRH, a central
regulator of reproduction, induces the UPR pathway through
activation of at least two of the three known ER stress sensors:
EIF2AK3, which leads to translation attenuation through phosphorylation of EIF2A, and ERN1, which leads to splicing of
transcription factor Xbp1 mRNA. Initial characterization of
UPR signal transduction comes from experiments using agents
that severely stress ER homeostasis and rapidly induce all three
arms of the UPR. However, evidence is mounting that the UPR
is crucial for normal physiological function, especially in the
development or function of secretory cells (12). The data presented here support this hypothesis and are the first to examine
activation of the UPR in gonadotropes.
Unlike pharmacological agents that severely disrupt ER function, physiological processes may be more subtle and utilize only
specific UPR components or activate the UPR to a lesser degree
or duration (12). This is observed in plasma cell differentiation,
which requires activation of ERN1 but not EIF2AK3 or EIF2A
phosphorylation (14). Loss of Eif2ak3 in mice results in loss of
␤-cell function (15), but loss of Xbp1 shows defects in liver
growth (41) and lymphocyte differentiation (13). In myocytes,
vasopressin at physiological concentrations activates the UPR
but not to the same degree as pharmacological agents. It also
does not result in chaperone synthesis or condition the cells to
tolerate future ER stressors (42), as pharmacological UPR activation has been shown to do. Whereas GNRH appears to target
EIF2AK3 and ERN1, it is unknown whether it targets ATF6.
ATF6 acts on ERSEs to stimulate transcription of Ddit3 (DNAdamage inducible transcript 3, also known as Chop or
Gadd153), Xbp1 (43) and chaperones, such as Hspa5 (heat
shock 70 kDa protein 5, also known as Grp78 or BiP), Hsp90b1
(heat shock protein 90 kDa ␤ member 1, also known as Grp94),
and Calr (calreticulin) (44). GNRH has been shown to induce
Xbp1 (19, 20) and Ddit3 (19). Induction of Xbp1 or Ddit3,
however, may also occur via XBP1 itself. Like ATF6, XBP1 can
bind to ERSEs, although with less affinity (45). Therefore,
GNRH may activate all three arms of the UPR, or just EIF2AK3
and ERN1, bypassing ATF6 and utilizing XBP1 instead to upregulate Ddit3 and its own mRNA. This remains to be investigated in gonadotropes.
The model presented in Fig. 9 illustrates that GNRH, through
activation of the IP3 receptor, causes calcium to leave the ER and
thus signals to activate EIF2AK3 and attenuate translation. The
involvement of intracellular calcium, but not other signal transducers of the known GNRH signaling pathways, is consistent with the
hypothesis that the loss of calcium from the ER elicits the UPR.
Ionomycin is an ionophore that carries Ca2⫹ ions across the
plasma membrane into the cytosol and facilitates release of calcium
from the ER, as demonstrated in a recent study (46). The data here
are consistent with data from others that show exposure of cells to
ionophores such as ionomycin inhibits translation and causes an
107
IP3
2.
Ca2+
ER
3.
4.
EIF2AK3
P
P
5.
EIF2A P
40S
6.
AA
A
FIG. 9. Proposed model for transient attenuation of translation induced by
GNRH. GNRH acting on its receptor (step 1) to induce calcium efflux from the ER
(step 2) leads to the loss of calcium and/or accumulation of unfolded proteins
(step 3) that allows dimerization and activation by autophosphorylation of
EIF2AK3 (step 4). Activated EIF2AK3 phosphorylates translation initiator EIF2A
(step 5), leading to translation attenuation through inhibition of initiation
complex formation and binding to mRNA (step 6). GNRHR, GNRH receptor; PLC,
phospholipase C; AAA, poly-adenylate tail.
increase in monosomes and free ribosomal subunits (42, 47). How
loss of calcium mechanistically causes activation of EIF2AK3 is
unclear. Multiple signals may integrate to activate the UPR. First,
the stimulus to secrete (␤-cells) or differentiate (B cells) may be
accompanied by an increase in demand for protein synthesis that
the ER is not yet prepared for, resulting in accumulation of misfolded proteins that activate the UPR (12). The ER-resident chaperone HSPA5 associates with EIF2AK3 and ERN1 to prevent their
dimerization and activation. Dissociation of HSPA5 from these
sensors caused by HSPA5 association with unfolded proteins during stress allows EIF2AK3 and ERN1 dimerization and activation
(23). Unfolded proteins may also bind directly to ERN1 or
EIF2AK3 (7). Data showing that cycloheximide rescues tunicamycin- or thapsigargin-induced impairment of cell growth in cells
lacking EIF2AK3 (48) are consistent with the idea that misfolded
proteins trigger the UPR. Second, secretory protein production and
secretion results in acute loss of amino acids and continued production of reactive oxygen species (ROS) generated by disulfide
bond formation. The ER may act as a sensor to combat oxidative
stress caused by accumulated ROS (49, 50). Cells lacking UPR
components accumulate higher levels of ROS that contribute to cell
death (51). Finally, calcium may be the primary integrator of stress.
Loss of calcium from the ER lumen during secretion may disrupt
folding of proteins that require calcium to fold properly, particularly glycoproteins (42). Calcium loss may also directly disrupt
chaperones, such as HSPA5, which bind calcium and are involved
in maintaining ER calcium levels (52). EIF2AK3 itself has been
implicated in integrating calcium-mediated ER stress responses
(26). Loss of ER calcium has been linked to UPR activation in
myocytes upon vasopressin stimulation (42). It is of interest to
determine the mechanism of EIF2AK3 activation, especially in the
108
Do et al.
GNRH Induces the UPR
context of physiological processes such as secretion. Calcium facilitates secretion (4) and transcriptional responses (53) in gonadotropes, and now it appears to play a role in translational control,
giving greater credence to its importance in normal pituitary
function.
GNRH regulates several posttranscriptional processes.
GNRH induces Lhb and Cga mRNA 3⬘-polyadenylation, leading to increased message stability (54). In the presence of
GNRH, Cga message half-life increases from 1.2 h to 8 h (55).
Interestingly, microarray data in HeLa cells show that mRNAs
that are translationally attenuated during ER stress are also
stabilized (56). This is consistent with the known increase in
stability and now observed translational attenuation of Lhb and
Cga mRNAs upon GNRH exposure. The receptor for GNRH
(Gnrhr) may also be translationally regulated. GNRH receptor
is down-regulated with exposure to high concentrations of
GNRH, and its mRNA is bound by fewer ribosomes without a
change in overall mRNA levels (57). Recent evidence specifically
addresses the regulation of translation by GNRH through examination of cap-binding initiation factors. GNRH causes inactivation of EIF4EBP1, a negative regulator of the EIF4E capbinding protein required for cap-dependent protein synthesis
(32, 58). In L␤T2 cells, GNRH induces activation (phosphorylation) of several cap-dependent translation initiation factors
(32). The stimulation of translation initiation factors can be
reconciled with a UPR to attenuate translation. First, whereas
the activation or increased availability of initiation factors may
serve to contribute to translation of transcripts, the UPR may
play an important role in regulating the fidelity of their translation. Second, although this remains to be addressed, the translation attenuation induced by GNRH may not result in attenuation of all mRNAs, because Xbp1 and Atf4 translation is
induced by the UPR in other systems. The translation of a particular mRNA during conditions of global regulation such as the
UPR may depend on the translational efficiency of that mRNA,
which in turn is determined by its abundance, structure, or sequence in untranslated regions (59). Coupling this with changes
in activation status, availability of initiation factors or other
trans-acting factors may provide a balance that determines
which mRNAs are sensitive to translation attenuation or
changes in the general translational machinery. This type of
specificity has been characterized in the well-studied stimulation
of translation of Atf4 mRNA (9) and the related yeast gene
GCN4 (60) during the UPR. In yeast, under conditions of general translational inhibition due to glucose deprivation (61),
amino acid deprivation (62), or rapamycin-induced stress (63),
various transcripts sediment with heavier polysomes, increasing
in translational efficiency. The activation of cap-binding initiation factors by GNRH within the context of the UPR may lend
specificity to the translational response.
It remains unknown what function the UPR serves in GNRH
action. On one hand, activation of the UPR could be considered
an acute developmental response to adapt the capacity of the ER
of gonadotropes. In culture, the L␤T2 cells can be considered
naïve to GNRH, and activation of the UPR may be necessary to
activate expansion of the ER, as is the case in plasma cells (10).
Recently, generation of ␤-cell-specific Eif2ak3 knockout mice
Mol Endocrinol, January 2009, 23(1):100 –112
has led to the hypothesis that Eif2ak3 is critical for ␤-cell proliferation and differentiation during fetal development, rather
than for preventing overburdening of the ER and apoptosis
postnatally (64). The requirement of Eif2ak3 and the UPR for
development of the mature gonadotrope may not be the case
here, however, because the primary pituitary cells used in this
study were from sexually mature mice that have already experienced GNRH in vivo and yet still up-regulate a marker of the
UPR upon GNRH exposure in culture.
On the other hand, the UPR may serve to regulate translational fidelity and protect gonadotropes from apoptosis. The
protective effect of the UPR has been observed in other secretory
cell types. Global knockout of Eif2ak3 (15, 16) or of the ability
to phosphorylate EIF2A (17) in mice leads to pancreatic ␤-cell
loss and the development of diabetes. Other secretory cells such
as hepatocytes and osteoblasts also show defects with the loss of
Eif2ak3 or other UPR components (12). Induction of the UPR
protects cells from injury by future stress (65, 66). This would fit
in well with the pulsatile action of GNRH. As measured in rats
(67) and sheep (68), GNRH is released once every 30 min to 1 h,
and pulses last for less than 10 min. Whereas perturbations in
the ER by pharmacological agents can be categorized as acute
insults, physiological processes such periodic hormonal input
for secretion can be viewed as chronic ER stress. Activation of
the UPR by GNRH is transient in L␤T2 cells and is induced at a
lower magnitude than by DTT. This is consistent with the idea
that GNRH is not activating an acute, extreme stress response
but rather modulating the level of protein production in the ER
to match synthetic capacity, adapting to physiological ER stress.
Similarly, myocytes rapidly recover protein synthesis after vasopressin stimulation of the UPR (42). Paradoxically, the UPR
can also signal to apoptotic pathways, such as through induction of Ddit3, which is thought to be induced by all three arms
of the UPR (8). GNRH highly induces Atf3 (18 –20), an inhibitor of Ddit3 (69). Microarray studies using various concentrations and durations of GNRH treatment do not reveal Ddit3 as
a target of GNRH action, except in one study where Ddit3 was
induced when chronic GNRH was given in conjunction with
activin (19). It is possible that Atf3 prevents induction of Ddit3,
thus conferring protection from apoptosis. In agreement with
this, whereas pharmacological exposure of L␤T2 cells to
GNRH agonist for 96 h increases apoptosis (70), GNRH has
been shown to be protective in vivo (71). Thus, the UPR might
serve to prevent or limit the degree of apoptosis, both from
GNRH as well as from the apoptotic signals that are induced by
the UPR itself.
Although GNRH transiently attenuates translation, pulselabeling experiments have not shown general changes in protein
synthesis in response to GNRH treatment (data not shown).
Steady-state levels of Lhb mRNA increases approximately 1.5fold with GNRH stimulation (72), and this may serve to counter
the 2-fold increase of Lhb mRNA in RNP complexes. Any observed decreases in overall translation may also be balanced by
stimulation of translation of mRNAs not explored in this study.
The transitory nature of translational attenuation induced by
GNRH via the UPR is not necessarily targeted at reducing overall protein synthesis, but rather at maintaining the fidelity of
Mol Endocrinol, January 2009, 23(1):100 –112
translation. Gonadotrope-specific loss of components of the
UPR in vivo may result in apoptotic loss of gonadotropes or loss
of LHB production due to increased levels of protein misfolding
and degradation, similar to the case described for islet cells (15).
Future studies using in vivo models of UPR disruption will be
necessary to fully characterize the physiological role of the UPR
in gonadotropin synthesis and secretion.
The data here show that GNRH, a neuropeptide hormone
that regulates reproduction, utilizes a regulatory mechanism
previously associated with starvation or stress, and more recently shown to be important for normal secretory function, to
modulate gene expression. GNRH signals to the UPR to target
the translation of specific mRNAs important to differentiated
gonadotrope cell function. This supports the idea that signaling
to the UPR by ER-disrupting agents can be thought of as an
extension of the UPR’s physiological role in sensing calcium and
maintaining the integrity of the ER and the quality of proteins
leaving it. This study leads to a more complete understanding of
the integration of multiple levels of gene regulation by GNRH
and adds to the physiological relevance of the UPR in maintaining cellular homeostasis.
Materials and Methods
L␤T2 and primary pituitary cell culture
The L␤T2 (3) mouse gonadotrope line was maintained in high-glucose HEPES-buffered DMEM supplemented with penicillin/streptomycin and 10% fetal bovine serum. The cells were incubated at 37 C in a
humidified atmosphere of 5% CO2.
For primary pituitary culture, pituitaries were isolated from 9-wkold wild-type male mice (C57BL/6). Mice were killed using CO2 asphyxiation followed by cervical dislocation, in accordance with UCSD Institutional Animal Care and Use Committee regulations. Whole pituitaries
were isolated into ice-cold PBS and then dissociated through incubation
in trypsin containing collagenase in a shaking 37 C bath for 15 min.
Afterward, the pituitaries were gently pipetted to facilitate dissociation
and then returned to the bath for another 15 min. Finally, deoxyribonuclease was added for 10 min, and then the cells were pelleted at
2000 ⫻ g for 5 min. The pelleted cells were resuspended and plated on
polylysine-coated dishes in the same conditions described for the L␤T2
cells.
Hormone and drug treatments
Cells were plated, grown overnight for 36 h before the media was
changed to DMEM without serum, and then incubated for an additional
16 –18 h. Cells were then treated with vehicle, 10 nM GNRH, 1 ␮M
ionomycin (Calbiochem, La Jolla, CA) for 30 min, 50 nM thapsigargin
for 1 h, 75 ␮M 2-APB (Calbiochem) for 10 min, 50 ␮M BAPTA (Calbiochem) for 30 min, 2 mM DTT for 30 min, or 10 nM EGF for 30 min.
Where appropriate, 5 ␮M actinomycin D (Calbiochem) was added to the
media for 1 h, 100 ␮g/ml cycloheximide for 5 min, or 1 ␮M U0126
(Calbiochem) for 30 min, before GNRH treatment. All reagents were
from Sigma-Aldrich (St. Louis, MO) unless otherwise indicated. GNRH
at 10 nM was previously shown to induce calcium flux and exocytosis in
L␤T2 (4). The drugs/hormones were left in the media for the duration of
treatment. For the GNRH time course, GNRH was added to the media
for 10 min, after which the media were removed, the cells were washed
with PBS, and fresh media were added back until harvest.
RNA isolation and RT-PCR
L␤T2 and primary pituitary cells were plated at a density of 1 ⫻ 107
cells per 10-cm dish or 300,000 cells per 48-well plate, respectively.
mend.endojournals.org
109
Total RNA was harvested using Trizol LS (Invitrogen, Carlsbad, CA).
The RNA was deoxyribonuclease treated and then reverse transcribed
using QuantiTect Reverse Transcription Kit (QIAGEN, Valencia, CA).
PCR was performed using HotStarTaq Mastermix (QIAGEN) and transcript-specific primers, as outlined below. Parallel control PCRs were
performed using all components except for reverse transcriptase. PCR
products were subjected to agarose electrophoresis in the presence of
ethidium bromide.
Primer sequences were designed against murine mRNA sequences as
available through PubMed. The sequences of the primers used were as
follows. Atf6 forward (fwd), 5⬘-AAAGTCCCAAGTCCAAAGC-3⬘;
Atf6 reverse (rev), 5⬘-CTGAAAATTCCAAGAGATGC-3⬘; Ern1 fwd,
5⬘-AACTCCTCTGTCTGCATCC-3⬘; Ern1 rev, 5⬘-GCCAACTATGTTGATAACTTCC-3⬘; Eif2ak3 fwd, 5⬘-AGCACGCAGATCACAGTCAGG-3⬘; Eif2ak3 rev, 5⬘-TGGCTACGATGCAAAGCAGG-3⬘.
Ribosome fractionation and mRNA isolation
Two 10-cm plates were used for each experimental condition with
2 ⫻ 107 L␤T2 cells seeded per plate and treated as described. After
treatment, 100 ␮g/ml cycloheximide was added to all the plates (except
for any plates already pretreated with cycloheximide), and the cells were
incubated on ice for 5 min. The cells were then harvested in 500 ␮l of
ice-cold polysome extraction buffer (PEB) containing 140 mM KCl, 5
mM MgCl2, 1 mg/ml heparin sodium salt, and 20 mM Tris-HCl (pH 8),
supplemented with fresh 0.5 mM DTT and 100 ␮g/ml cycloheximide on
the day of the experiment. Cells from two plates of the same experimental condition were collected and pooled at this step. The cells were spun
down at 1000 ⫻ g for 3 min, and then each pellet was resuspended in
200 ␮l PEB containing 1% Triton-X. The pellets were allowed to lyse on
ice for 20 min with gentle inversion by hand every few minutes. Finally,
the cellular debris was collected by centrifugation at 10,000 ⫻ g for 10
min. The supernatants were layered onto 5 ml 10 –50% sucrose gradients made with PEB lacking Triton-X. The samples were centrifuged in
a SW-55 swing-bucket rotor for 1.5 h at 150,000 ⫻ g.
A 21-gauge needle was used to collect fractions from the bottom of
the gradient. Fractions were collected using a peristaltic pump while
monitoring real-time absorption at 254 nm through a UVM-II monitor
(GE Healthcare, Fairfield, CT). Fractions of 250 ␮l volume each were
collected using a FC 203B fraction collector (Gilson, Middleton, WI).
Monitoring of UV absorption and fraction collection was controlled
through a Labview virtual instrument (National Instruments, Austin,
TX) and a KPCI 3108 DAQ card (Keithley Instruments, Cleveland,
OH). Fractions were dripped directly into sodium dodecyl sulfate for a
final sodium dodecyl sulfate concentration of 1%. Each fraction was
then treated with 0.2 mg/ml proteinase K at 37 C for 1.5 h.
RNA was purified from each fraction by phenol-chloroform extraction and ethanol precipitation. An aliquot (1 ␮l) of RNA from each
fraction was subjected to agarose electrophoresis in the presence of
ethidium bromide to verify the presence of the large and small ribosomal
subunits through presence of the 28S and 18S rRNAs, respectively. The
rest of the RNA from the fractions was pooled into polysome and RNP
pools according to the gradient’s UV absorption profile. PolyA RNA
was then isolated using Oligotex mRNA Mini Kit oligo-dT columns
(QIAGEN) according to the manufacturer’s recommendations and
eluted into a final volume of 40 ␮l for each pool. A 10 ␮l aliquot of
mRNA was used for reverse transcription for quantitative PCR analysis.
Quantitative real-time PCR
For quantitative PCR analysis of fractionated, ribosome-bound
mRNA, cDNA was synthesized using Omniscript Reverse Transcriptase
(QIAGEN) and 10 ␮M of random hexamer primers (Applied Biosystems, Foster City, CA). For analysis of primary pituitary RNA, cDNA
was synthesized using QuantiTect Reverse Transcription Kit (QIAGEN). Quantitative real-time PCR was carried out using the MyIQ
Single-Color Real-Time Detection System (Bio-Rad Laboratories, Hercules, CA). The PCR products were amplified in the presence of SYBR
Green using the QuantiTect SYBR Green PCR kit (QIAGEN) supplemented with 300 nM of transcript-specific primers and 1 ␮M fluorescein
110
Do et al.
GNRH Induces the UPR
Mol Endocrinol, January 2009, 23(1):100 –112
for proper instrument calibration. The cycling conditions used were
those recommended by the PCR kit manufacturer. A melt curve was
performed after each PCR run to ensure that just a single product was
amplified in each assay, and the size of the products was verified using
agarose gel electrophoresis.
Primer sequences were designed against murine mRNA sequences as
available through PubMed. The Xbp1s primer set only recognizes the
spliced Xbp1 mRNA. The primer sequences were as follows. Cga fwd,
5⬘-GGTTCCAAAGAATATTACCTCG-3⬘; Cga rev, 5⬘-GTCATTCTGGTCATGCTGTCC-3⬘; Gapdh fwd, 5⬘-TGCACCACCAACTGCTTAG-3⬘; Gapdh rev, 5⬘-GATGCAGGGATGATGTTC-3⬘; Lhb fwd,
5⬘-CTGTCAACGCAACTCTGG-3⬘; Lhb rev, 5⬘-ACAGGAGGCAAAGCAGC-3⬘; Xbp1s fwd, 5⬘-GAGTCCGCAGCAGGTG-3⬘; Xbp1s rev,
5⬘-GAATCTGAAGAGGCAACAGTG-3⬘.
Primers were designed to generate an 80 –150 bp amplicon that
crossed intron/exon boundaries where possible to avoid amplification of
any contaminating unspliced RNA or genomic DNA. All PCRs were
performed in triplicate, except for those involving cDNA from primary
pituitary cells, which were performed in duplicate. A larger cDNA fragment of Cga, Gapdh, and Lhb was inserted into pCR2.1 (Invitrogen)
and used to generate a standard curve and assess PCR efficiency for each
run. The mass values for each transcript were extrapolated from the
standard curve. For Xbp1s PCRs, cDNA from DTT-treated L␤T2 cells
were diluted serially and run alongside primary pituitary reactions to
assess efficiency. In this case, the Pfaffl method (73) was used to calculate relative mass levels of Xbp1s with respect to Gapdh as an internal
control.
To calculate a fold-redistribution value for an mRNA’s movement
within the ribosome profile, the mass of each transcript in each ribosome
pool (polysome or RNP) was measured, and a ratio was calculated for
the mass of that transcript in the RNP pool compared with the polysome
pool. A ratio was calculated for both the treated and control (vehicle
treated) conditions. A fold-redistribution value was then generated by
taking a ratio of the ratios: the RNP/polysome ratio in treated compared
with the RNP/polysome ratio in control cells. Statistics were performed
as indicated in each figure legend on these values. For ease of representation in the histograms only, the negative inverse was calculated and
reported for fold-redistribution values between 0 and 1, such that movement of an mRNA into the polysome pool would report a negative
redistribution value, and movement into RNP complexes would report
a positive value.
The areas under the curves of the ribosome profiles were integrated
using SigmaPlot version 9.01 (Systat Software, Richmond, CA). Any
ratio or fold-redistribution data were transformed using log2, and a
value of 10 was added to those values to ensure positive values before
analysis. All subsequent analysis was conducted using JMP (SAS Institute, Cary, NC) on untransformed (other than log2 ⫹ 10 for ratio or
fold-redistribution values) or optimally Box-Cox transformed values.
All experiments were repeated at least three independent times, and
reported values are the means ⫾ SEM.
Xbp1 splicing assay
References
Xbp1 splicing was detected through PCR amplification of Xbp1 by
primers that recognize both the spliced and unspliced mRNA, followed
by PstI digestion. The primers used were as follows: fwd, 5⬘-TACGGGAGAAAACTCACG-3⬘; rev, 5⬘-TCTGAAGAGCTTAGAGGTGC-3⬘.
After PCR amplification, Xbp1 cDNA (10 ␮l of a 50 ␮l PCR) was
treated with 20 – 40 U of PstI (New England BioLabs, Ipswich, MA) for
1 h and then resolved by agarose electrophoresis in the presence of
ethidium bromide. The expected PCR band sizes were 549 bp for the
unspliced and 523 bp for the spliced form. PstI digestion of unspliced
Xbp1 yields fragments of 172 and 381 bp whereas the spliced mRNA is
resistant to PstI.
Western blotting
Cells were plated (1 ⫻ 107) in 10-cm dishes. After treatment, protein
was harvested using standard radioimmune precipitation assay lysis
buffer supplemented with phosphatase inhibitors. Standard SDS-PAGE
and semidry transfer method (Bio-Rad Laboratories, Hercules, CA) was
used to transfer the extracts onto polyvinylidenedifluoride membranes
(Bio-Rad Laboratories). Species-specific antibodies were used for all
subsequent blotting.
For detecting phosphorylated and total EIF2A, membranes were
blocked with 5% nonfat dry milk and the primary antibodies (Cell
SignalingTechnology, Inc., Danvers, MA) delivered in 5% BSA. For
EIF2AK3, blocking and primary antibody (Rockland Immunochemicals, Gilbertsville, PA) delivery was performed in 5% nonfat dry milk.
For detecting phosphorylated ERK, blocking and primary antibody
(Santa Cruz Biotechnology, Inc., Santa Cruz, CA) delivery was performed in 1⫻ casein (Vector Laboratories, Burlingame, CA). All primary antibodies were diluted 1:1000 and incubated overnight. Blots
were developed using 1:2000 dilution of biotinylated secondary antibodies (Santa Cruz Biotechnology) and Chemiglow chemiluminescence
(Alpha Innotech, San Leandro, CA). Chemiluminescence was visualized
using the GeneSnap BioImaging System (Syngene, Frederick, MD).
For confirmation of the phosphorylation status of EIF2AK3, extracts were lysed in radioimmune precipitation assay buffer supplemented with protease inhibitors (Roche, Basel, Switzerland) but not
phosphatase inhibitors. Extracts were then treated with 2000 U of ␭
Protein Phosphatase (New England BioLabs, Ipswich, MA) for 15 min
before SDS-PAGE.
Statistical analysis
Acknowledgments
We thank Lisa Gallegos for performing the fluorescence resonance energy transfer assays to assess PKC activation.
Address all correspondence and requests for reprints to: Mark A. Lawson, Department of Reproductive Medicine, Mail Code 0674, University of
California, San Diego, La Jolla, California 92093-0674. E-mail: mlawson@
ucsd.edu.
This work was supported by National Institutes of Health Grants
R01 HD043758, K02 HD040803, and U54 HD12303 (to M.L) and
Grant T32 GM008666 (to M.T.D.).
Disclosure Statement: The authors have nothing to disclose.
1. Pawson AJ, McNeilly AS 2005 The pituitary effects of GNRH. Anim Reprod
Sci 88:75–94
2. Burger LL, Haisenleder DJ, Dalkin AC, Marshall JC 2004 Regulation of
gonadotropin subunit gene transcription. J Mol Endocrinol 33:559 –584
3. Alarid ET, Windle JJ, Whyte DB, Mellon PL 1996 Immortalization of pituitary cells at discrete stages of development by directed oncogenesis in transgenic mice. Development 122:3319 –3329
4. Thomas P, Mellon PL, Turgeon JL, Waring DW 1996 The LbT2 clonal
gonadotrope: a model for single cell studies of endocrine cell secretion. Endocrinology 137:2979 –2989
5. Kraus S, Naor Z, Seger R 2001 Intracellular signaling pathways mediated by
the gonadotropin-releasing hormone (GNRH) receptor. Arch Med Res 32:
499 –509
6. Kaiser UB, Conn PM, Chin WW 1997 Studies of gonadotropin-releasing
hormone (GNRH) action using GNRH receptor-expressing pituitary cell
lines. Endocr Rev 18:46 –70
7. Ron D, Walter P 2007 Signal integration in the endoplasmic reticulum unfolded protein response. Nat Rev Mol Cell Biol 8:519 –529
8. Schroder M, Kaufman RJ 2006 Divergent roles of IRE1␣ and PERK in the
unfolded protein response. Curr Mol Med 6:5–36
9. Kaufman RJ 2004 Regulation of mRNA translation by protein folding in the
endoplasmic reticulum. Trends Biochem Sci 29:152–158
10. Rutkowski DT, Kaufman RJ 2004 A trip to the ER: coping with stress. Trends
Cell Biol 14:20 –28
11. Kang SW, Rane NS, Kim SJ, Garrison JL, Taunton J, Hegde RS 2006 Substrate-specific translocational attenuation during ER stress defines a pre-emptive quality control pathway. Cell 127:999 –1013
Mol Endocrinol, January 2009, 23(1):100 –112
12. Wu J, Kaufman RJ 2006 From acute ER stress to physiological roles of the
unfolded protein response. Cell Death Differ 13:374 –384
13. Reimold AM, Iwakoshi NN, Manis J, Vallabhajosyula P, Szomolanyi-Tsuda
E, Gravallese EM, Friend D, Grusby MJ, Alt F, Glimcher LH 2001 Plasma cell
differentiation requires the transcription factor XBP-1. Nature 412:300 –307
14. Zhang K, Wong HN, Song B, Miller CN, Scheuner D, Kaufman RJ 2005 The
unfolded protein response sensor IRE1␣ is required at 2 distinct steps in B cell
lymphopoiesis. J Clin Invest 115:268 –281
15. Harding HP, Zeng H, Zhang Y, Jungries R, Chung P, Plesken H, Sabatini DD,
Ron D 2001 Diabetes mellitus and exocrine pancreatic dysfunction in
perk⫺/⫺ mice reveals a role for translational control in secretory cell survival.
Mol Cell 7:1153–1163
16. Zhang P, McGrath B, Li S, Frank A, Zambito F, Reinert J, Gannon M, Ma K,
McNaughton K, Cavener DR 2002 The PERK eukaryotic initiation factor 2 ␣
kinase is required for the development of the skeletal system, postnatal
growth, and the function and viability of the pancreas. Mol Cell Biol 22:
3864 –3874
17. Scheuner D, Song B, McEwen E, Liu C, Laybutt R, Gillespie P, Saunders T,
Bonner-Weir S, Kaufman RJ 2001 Translational control is required for the
unfolded protein response and in vivo glucose homeostasis. Mol Cell 7:1165–
1176
18. Kakar SS, Winters SJ, Zacharias W, Miller DM, Flynn S 2003 Identification of
distinct gene expression profiles associated with treatment of L␤T2 cells with
gonadotropin-releasing hormone agonist using microarray analysis. Gene
308:67–77
19. Zhang H, Bailey JS, Coss D, Lin B, Tsutsumi R, Lawson MA, Mellon PL,
Webster NJ 2006 Activin modulates the transcriptional response of L␤T2
cells to GNRH and alters cellular proliferation. Mol Endocrinol 20:2909 –
2930
20. Lawson MA, Tsutsumi R, Zhang H, Talukdar I, Butler BK, Santos SJ, Mellon
PL, Webster NJ 2007 Pulse sensitivity of the luteinizing hormone ␤ promoter
is determined by a negative feedback loop Involving early growth response-1
and Ngfi-A binding protein 1 and 2. Mol Endocrinol 21:1175–1191
21. Jiang HY, Wek SA, McGrath BC, Lu D, Hai T, Harding HP, Wang X, Ron D,
Cavener DR, Wek RC 2004 Activating transcription factor 3 is integral to the
eukaryotic initiation factor 2 kinase stress response. Mol Cell Biol 24:1365–
1377
22. Lee AH, Iwakoshi NN, Glimcher LH 2003 XBP-1 regulates a subset of endoplasmic reticulum resident chaperone genes in the unfolded protein response. Mol Cell Biol 23:7448 –7459
23. Bertolotti A, Zhang Y, Hendershot LM, Harding HP, Ron D 2000 Dynamic
interaction of BiP and ER stress transducers in the unfolded-protein response.
Nat Cell Biol 2:326 –332
24. Ma Y, Lu Y, Zeng H, Ron D, Mo W, Neubert TA 2001 Characterization of
phosphopeptides from protein digests using matrix-assisted laser desorption/
ionization time-of-flight mass spectrometry and nanoelectrospray quadrupole
time-of-flight mass spectrometry. Rapid Commun Mass Spectrom 15:1693–
1700
25. Hollien J, Weissman JS 2006 Decay of endoplasmic reticulum-localized
mRNAs during the unfolded protein response. Science 313:104 –107
26. Liang SH, Zhang W, McGrath BC, Zhang P, Cavener DR 2006 PERK (eIF2␣
kinase) is required to activate the stress-activated MAPKs and induce the
expression of immediate-early genes upon disruption of ER calcium homoeostasis. Biochem J 393:201–209
27. Preiss T, M WH 2003 Starting the protein synthesis machine: eukaryotic
translation initiation. Bioessays 25:1201–1211
28. Yoshida H 2007 Unconventional splicing of XBP-1 mRNA in the unfolded
protein response. Antioxid Redox Signal 9:232323–232333
29. Hirota M, Kitagaki M, Itagaki H, Aiba S 2006 Quantitative measurement of
spliced XBP1 mRNA as an indicator of endoplasmic reticulum stress. J Toxicol Sci 31:149 –156
30. Rispoli LA, Nett TM 2005 Pituitary gonadotropin-releasing hormone
(GNRH) receptor: structure, distribution and regulation of expression. Anim
Reprod Sci 88:57–74
31. Thomas G, Martin-Perez J, Siegmann M, Otto AM 1982 The effect of serum,
EGF, PGF2 ␣ and insulin on S6 phosphorylation and the initiation of protein
and DNA synthesis. Cell 30:235–242
32. Nguyen KA, Santos SJ, Kreidel MK, Diaz AL, Rey R, Lawson MA 2004 Acute
regulation of translation initiation by gonadotropin-releasing hormone in the
gonadotrope cell line L␤T2. Mol Endocrinol 18:1301–1312
33. Dennis PB, Fumagalli S, Thomas G 1999 Target of rapamycin (TOR): balancing the opposing forces of protein synthesis and degradation. Curr Opin
Genet Dev 9:49 –54
34. Raught B, Gingras A-C, Sonenberg N 2000 Regulation of ribosomal recruitment in eucaryotes. In: Sonenberg N, Hershey JWB, Mathews M, eds. Trans-
mend.endojournals.org
35.
36.
37.
38.
39.
40.
41.
42.
43.
44.
45.
46.
47.
48.
49.
50.
51.
52.
53.
54.
55.
56.
57.
111
lational control of gene expression. 2nd ed. Cold Spring Harbor, NY: Cold
Spring Harbor Laboratory Press; 245–294
Rose A, Froment P, Perrot V, Quon MJ, LeRoith D, Dupont J 2004 The
luteinizing hormone-releasing hormone inhibits the anti-apoptotic activity of
insulin-like growth factor-1 in pituitary ␣T3 cells by protein kinase C␣-mediated negative regulation of Akt. J Biol Chem 279:52500 –52516
Mutiara S, Kanasaki H, Harada T, Oride A, Miyazaki K 2008 The involvement of phosphatidylinositol 3-kinase in gonadotropin-releasing hormoneinduced gonadotropin ␣- and FSH␤-subunit genes expression in clonal gonadotroph L␤T2 cells. Mol Cell Endocrinol 283:1–11
Liu F, Ruiz MS, Austin DA, Webster NJ 2005 Constitutively active Gq impairs gonadotropin-releasing hormone-induced intracellular signaling and luteinizing hormone secretion in L␤T2 cells. Mol Endocrinol 19:2074 –2085
Hendershot LM 2004 The ER function BiP is a master regulator of ER function. Mt Sinai J Med 71:289 –297
Yuen T, Wurmbach E, Ebersole BJ, Ruf F, Pfeffer RL, Sealfon SC 2002
Coupling of GNRH concentration and the GNRH receptor-activated gene
program. Mol Endocrinol 16:1145–1153
Wurmbach E, Yuen T, Ebersole BJ, Sealfon SC 2001 Gonadotropin-releasing
hormone receptor-coupled gene network organization. J Biol Chem
276:47195– 47201
Reimold AM, Etkin A, Clauss I, Perkins A, Friend DS, Zhang J, Horton HF,
Scott A, Orkin SH, Byrne MC, Grusby MJ, Glimcher LH 2000 An essential
role in liver development for transcription factor XBP-1. Genes Dev 14:152–
157
Brostrom MA, Brostrom CO 2003 Calcium dynamics and endoplasmic reticular function in the regulation of protein synthesis: implications for cell
growth and adaptability. Cell Calcium 34:345–363
Yoshida H, Okada T, Haze K, Yanagi H, Yura T, Negishi M, Mori K 2000
ATF6 activated by proteolysis binds in the presence of NF-Y (CBF) directly to
the cis-acting element responsible for the mammalian unfolded protein response. Mol Cell Biol 20:6755– 6767
Yoshida H, Haze K, Yanagi H, Yura T, Mori K 1998 Identification of the
cis-acting endoplasmic reticulum stress response element responsible for transcriptional induction of mammalian glucose-regulated proteins. Involvement
of basic leucine zipper transcription factors. J Biol Chem 273:33741–33749
Yamamoto K, Yoshida H, Kokame K, Kaufman RJ, Mori K 2004 Differential
contributions of ATF6 and XBP1 to the activation of endoplasmic reticulum
stress-responsive cis-acting elements ERSE, UPRE and ERSE-II. J Biochem
136:343–350
Elzi DJ, Bjornsen AJ, MacKenzie T, Wyman TH, Silliman CC 2001 Ionomycin causes activation of p38 and p42/44 mitogen-activated protein kinases in
human neutrophils. Am J Physiol Cell Physiol 281:C350 –C360
Brostrom CO, Chin KV, Wong WL, Cade C, Brostrom MA 1989 Inhibition
of translational initiation in eukaryotic cells by calcium ionophore. J Biol
Chem 264:1644 –1649
Harding HP, Zhang Y, Bertolotti A, Zeng H, Ron D 2000 Perk is essential for
translational regulation and cell survival during the unfolded protein response. Mol Cell 5:897–904
Rutkowski DT, Kaufman RJ 2003 All roads lead to ATF4. Dev Cell 4:442–
444
Tu BP, Weissman JS 2004 Oxidative protein folding in eukaryotes: mechanisms and consequences. J Cell Biol 164:341–346
Harding HP, Zhang Y, Zeng H, Novoa I, Lu PD, Calfon M, Sadri N, Yun C,
Popko B, Paules R, Stojdl DF, Bell JC, Hettmann T, Leiden JM, Ron D 2003
An integrated stress response regulates amino acid metabolism and resistance
to oxidative stress. Mol Cell 11:619 – 633
Lievremont JP, Rizzuto R, Hendershot L, Meldolesi J 1997 BiP, a major
chaperone protein of the endoplasmic reticulum lumen, plays a direct and
important role in the storage of the rapidly exchanging pool of Ca2⫹. J Biol
Chem 272:30873–30879
Jorgensen JS, Quirk CC, Nilson JH 2004 Multiple and overlapping combinatorial codes orchestrate hormonal responsiveness and dictate cell-specific expression of the genes encoding luteinizing hormone. Endocr Rev 25:521–542
Weiss J, Crowley Jr WF, Jameson JL 1992 Pulsatile gonadotropin-releasing
hormone modifies polyadenylation of gonadotropin subunit messenger ribonucleic acids. Endocrinology 130:415– 420
Chedrese PJ, Kay TW, Jameson JL 1994 Gonadotropin-releasing hormone
stimulates glycoprotein hormone ␣-subunit messenger ribonucleic acid
(mRNA) levels in ␣ T3 cells by increasing transcription and mRNA stability.
Endocrinology 134:2475–2481
Kawai T, Fan J, Mazan-Mamczarz K, Gorospe M 2004 Global mRNA stabilization preferentially linked to translational repression during the endoplasmic reticulum stress response. Mol Cell Biol 24:6773– 6787
Tsutsumi M, Laws SC, Rodic V, Sealfon SC 1995 Translational regulation of
112
58.
59.
60.
61.
62.
63.
64.
65.
Do et al.
GNRH Induces the UPR
the gonadotropin-releasing hormone receptor in ␣ T3-1 cells. Endocrinology
136:1128 –1136
Sosnowski R, Mellon PL, Lawson MA 2000 Activation of translation in
pituitary gonadotrope cells by gonadotropin-releasing hormone. Mol Endocrinol 14:1811–1819
Dever TE 2002 Gene-specific regulation by general translation factors. Cell
108:545–556
Gebauer F, Hentze MW 2004 Molecular mechanisms of translational control.
Nat Rev Mol Cell Biol 5:827– 835
Kuhn KM, DeRisi JL, Brown PO, Sarnow P 2001 Global and specific translational regulation in the genomic response of Saccharomyces cerevisiae to a
rapid transfer from a fermentable to a nonfermentable carbon source. Mol
Cell Biol 21:916 –927
Smirnova JB, Selley JN, Sanchez-Cabo F, Carroll K, Eddy AA, McCarthy JE,
Hubbard SJ, Pavitt GD, Grant CM, Ashe MP 2005 Global gene expression
profiling reveals widespread yet distinctive translational responses to different
eukaryotic translation initiation factor 2B-targeting stress pathways. Mol Cell
Biol 25:9340 –9349
Preiss T, Baron-Benhamou J, Ansorge W, Hentze MW 2003 Homodirectional changes in transcriptome composition and mRNA translation induced
by rapamycin and heat shock. Nat Struct Biol 10:1039 –1047
Zhang W, Feng D, Li Y, Iida K, McGrath B, Cavener DR 2006 PERK
EIF2AK3 control of pancreatic ␤ cell differentiation and proliferation is required for postnatal glucose homeostasis. Cell Metab 4:491– 497
Hung CC, Ichimura T, Stevens JL, Bonventre JV 2003 Protection of renal
epithelial cells against oxidative injury by endoplasmic reticulum stress pre-
Mol Endocrinol, January 2009, 23(1):100 –112
66.
67.
68.
69.
70.
71.
72.
73.
conditioning is mediated by ERK1/2 activation. J Biol Chem 278:29317–
29326
Lu PD, Jousse C, Marciniak SJ, Zhang Y, Novoa I, Scheuner D, Kaufman RJ,
Ron D, Harding HP 2004 Cytoprotection by pre-emptive conditional phosphorylation of translation initiation factor 2. EMBO J 23:169 –179
Sisk CL, Richardson HN, Chappell PE, Levine JE 2001 In vivo gonadotropinreleasing hormone secretion in female rats during peripubertal development
and on proestrus. Endocrinology 142:2929 –2936
Moenter SM, Brand RM, Midgley AR, Karsch FJ 1992 Dynamics of gonadotropin-releasing hormone release during a pulse. Endocrinology 130:503–
510
Wolfgang CD, Chen BP, Martindale JL, Holbrook NJ, Hai T 1997 gadd153/
Chop10, a potential target gene of the transcriptional repressor ATF3. Mol
Cell Biol 17:6700 – 6707
Miles LE, Hanyaloglu AC, Dromey JR, Pfleger KD, Eidne KA 2004 Gonadotropin-releasing hormone receptor-mediated growth suppression of immortalized L␤T2 gonadotrope and stable HEK293 cell lines. Endocrinology 145:
194 –204
Yin P, Arita J 2002 Proestrous surge of gonadotropin-releasing hormone
secretion inhibits apoptosis of anterior pituitary cells in cycling female rats.
Neuroendocrinology 76:272–282
Burger LL, Dalkin AC, Aylor KW, Haisenleder DJ, Marshall JC 2002 GNRH
pulse frequency modulation of gonadotropin subunit gene transcription in
normal gonadotropes-assessment by primary transcript assay provides evidence for roles of GNRH and follistatin. Endocrinology 143:3243–3249
Pfaffl MW 2001 A new mathematical model for relative quantification in
real-time RT-PCR. Nucleic Acids Res 29:e45